Abstract As the most important inhibitory neurotransmitter in the brain, a detailed understanding of the implications of gamma-aminobutyric acid (GABA) release remains elusive. The measurement of GABA concentrations is a difficult process. Microdialysis is the current standard for GABA sampling in the brains of freely moving animals, but suffers from low temporal resolution and the need for labor intensive analysis methods. By contrast, the direct sensing of GABA, by modalities including biosensors, would provide second-by-second temporal resolution, without the need for additional post- analysis. However, biosensors and other monitoring devices, require an enzyme to process the analyte of interest. The state-of-the-art for the enzymatic conversion of GABA into a transducible signal is the sequential activity of multiple enzymes or antibodies entrapped within nanoparticles. For brain and systemic GABA sensing applications, a single GABA oxidase enzyme is necessary. No such oxidase enzyme for GABA is currently available. This proposal addresses this problem by designing a single GABA-specific oxidase enzyme for use as the biorecognition element that is suitable for fabrication of a GABA biosensor. During Phase I we identified, isolated, purified, cloned, crystallized and modeled the active-site structure of ?-N- Methylaminobutyrate oxidase (MGOX). We set up a directed evolution system (KJ109) in which there will only be growth if GABA is available as a nitrogen source, and we demonstrated that the preliminary MGOX enzyme can be used on a biosensor to detect GABA. For Phase II of this project, there are two major problems that must be solved: (1) Modification of MGOX to increase its kcat at physiological pH (~pH 7.4); and (2) Modification of MGOX to increase sensitivity to detect physiologically relevant GABA concentrations. Pinnacle will team with an interdisciplinary group of two leading scientists at the University of Kansas. Professor Mark Richter, is an expert in protein engineering and protein folding, and Dr. Philip Gao, is the Director of the Protein Production Core Facility. By the end of Phase II, two commercially available products will be available. First, a GABA biosensor for real-time measurement of physiologically relevant levels of GABA in the brain for preclinical animal models and second, a GABA oxidase enzyme for use in a variety of diagnostic and point-of-care devices.